This proposal aims to use the leukocyte integrin CD11a (leukocyte function antigen-1, LFA-1) promoter to direct tissue and stage specific expression of reporter genes in transgenic mice. The integrin gene family codes for heterodimers which mediate cell to cell and cell to matrix adherence. CDI Ia belongs to the beta-2 subclass, or leukocyte, integrins, which mediate cellular adherence in inflammation and the immune response. Leukocyte integrins are responsible for leukocyte margination and emigration into inflamed tissues, and function as co-receptors in tumor and microbial killing and T-cell activation. The leukocyte integrin molecules consist of one of three alpha chains (CD11a, CD11b, or CD11c) noncovalently bound to a single common beta chain (CD18) to form three different heterodimers. Although CD18 is expressed on all leukocytes, CD11a is expressed on T and B lymphocytes, monocytes, and macrophages. The leukocyte integrins are also differentially expressed in leukocyte ontogeny, showing, in general, increased expression with leukocyte maturation, differentiation, and activation. In vitro transfection studies of CD11a-promoter -human growth hormone reporter gene constructs have defined regions of the CD11a promoter responsible for high level, tissue specific expression.The experiments described in this proposal will investigate the regulation of the CD11a promoter in transgenic mice. A construct consisting of 0.8 kilobases of the human CD11a promoter driving a human CD4-human growth hormone minigene is currently being injected into mouse oocytes. The cytoplasmic portion of the CD4 gene has been deleted to reduce the possibility of intracellular signalling via associated protein kinases so that CD4 may serve only as a cell surface reporter molecule. Initial experiments will detect the integrated transgene and characterize its expression. Lineage specific expression on leukocytes will be assayed by FACS analysis of peripheral blood and lymph node leukocytes . Tissue specific expression will be further characterized by screening various tissues for transgene mRNA using RNA blots and a reverse transcriptase-PCR assay. Additional regulatory sequences will be delineated by injecting other constructs having additional 5', intronic, and 3' sequences from the human CD11a gene . This approach, plus a comparison with parallel transgenic studies using the human CD11b and CD18 promoters should delineate regulatory regions responsible for differential gene expression in lymphocytes and monocytes/macrophages and provide insight into the sequences responsible for coordinate regulation of the CD11 and CD18 subunits during hematopoietic cell differentiation. Long-term plans involve using the CD11a promoter to: 1) express inducible cellular toxins in leukocytes to create an animal model in which to study the role of leukocytes in inflammation; and 2) express selected oncogenes in leukocytes to study the differentiation of B cells and monocytes/macrophages. In sum, these experiments will investigate the regulation of a)11a, an important leukocyte adherence molecule, and use the CD11a promoter in transgenic mice to study leukocyte function and ontogeny.
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